The season may affect the values of fresh semen variables and therefore influence the success of cryopreservation. The aim of this study was to improve the evaluation of seasonal changes in semen quality in Spanish Black Castellana roosters maintained under natural environmental conditions. Semen was collected from 11 Black Castellana roosters (housed under natural photoperiod and temperature conditions) by massage twice every month for 12 mo. In addition to determining ejaculate volume, sperm concentration, and sperm motility (the classic sperm variables), we used the hypo-osmotic swelling test to examine the membrane integrity of the spermatozoa. Further, morphological abnormalities and acrosome integrity were assessed via aniline blue staining. Semen volume (P < 0.05), sperm concentration (P < 0.01), and the percentage of spermatozoa with an intact acrosome (P < 0.01) were significantly affected by the season of the year. The annual profile of the percentage of spermatozoa showing acrosome integrity followed a trend roughly parallel to annual variations in temperature (Spearman rank correlation = 0.77, P < 0.01). According to the hypo-osmotic swelling test, membrane integrity fell in July (P < 0.05 compared with all other months), the month of highest temperatures. Aniline blue staining and the hypo-osmotic swelling test provide an easy and useful means of evaluating sperm abnormalities, including acrosome morphology and membrane integrity, and could be easily introduced into routine avian semen quality assessments. The results show that high semen quality is associated with long day photoperiods. Extreme heat or cold appear to exert a negative influence on sperm quality.

A sperm cryopreservation protocol requiring dimethylacetamide (DMA, 6%) as a cryoprotectant was optimized via assays involving different prefreezing equilibration times (1, 10, 30, 60, and 120 min at 5°C) and different freezing rates achieved by the following: 1) using nitrogen vapor to reduce the temperature from 5°C to -85°C at 10°C/min (slow freezing rate); 2) using a biological freezer unit in a 2-step method to reduce the temperature from 5°C to -35°C at 7°C/min and then from -35°C to -140°C at 60°C/min (medium freezing rate); or 3) using a biological freezer unit in a 1-step freezing method to reduce the temperature from 5°C to -180°C at 60°C/min (rapid freezing rate). Heterospermic semen samples from chicken breeds raised as part of a Spanish genetic resource conservation program were used in all assays. The 1-min equilibration treatment was associated with a lower percentage of viable thawed spermatozoa than the 30-min treatment (P < 0.05). The remaining sperm variables studied were not affected by equilibration time. The medium-rate 2-step freezing method was associated with a higher percentage of motile spermatozoa after thawing and with greater acrosome integrity (P < 0.05) than the slow nitrogen vapor or rapid 1-step methods. Thawed sperm movement quality and plasma membrane integrity (as assessed by the hypoosmotic swelling test) were better (P < 0.05) in samples frozen by the medium-rate 2-step freezing method than in those subjected to the slow nitrogen vapor method. Fertility was not influenced by freezing method, although that achieved with the medium rate 2-step freezing method showed a trend toward being greater than that achieved with the rapid 1-step method (P = 0.07). Together, the present results suggest that slow cooling rates are not recommendable when using dimethylacetamide. The 2-step freezing method may be useful in the establishment of a germplasm bank for Spanish chicken breeds.

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat (‘good freezer’) and Black-Red Andaluza (‘bad freezer’) showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages.

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This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre-freezing equilibration times (2-3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60-85°C min ), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified-warmed sperm variables were at their best when the spermatozoa was diluted in TCG-6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.

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This work examines the effect of the interaction between different concentrations of two cryoprotectants - glycerol (GLY) and dimethylacetamide (DMA) - and two methods of cryopreservation - pellets produced by plunging into liquid nitrogen and gradual in-straw freezing - on frozen/thawed chicken sperm variables. Sperm was cryopreserved using: (i) 6% DMA, following the in-straw and the pellet methods (ii) 11% GLY, following the in-straw and the pellet methods; and (iii) 8% GLY in the in-straw method and 3% DMA in the pellet method (i.e. reduced cryoprotectant concentrations). When 6% DMA was used as the cryoprotectant, no differences were seen between the in-straw and pellet methods in terms of frozen/thawed sperm variables or fertility (10.8% and 12.8%, respectively). The viability and motility variables of the frozen/thawed sperm produced using the in-straw method with 11% GLY were higher (p < 0.05) than those recorded for the sperm preserved using the same cryoprotectant and concentration in the pellet method. However, fertility was extremely low in both groups (2.1% and 4.2% for the in-straw and pellet methods, respectively). Finally, the use of 8% GLY in the in-straw method returned higher sperm viability, intact acrosome and motility values than the use of 3% DMA in the pellet method (p < 0.01). No differences were seen, however, in the fertility results obtained (28.8% and 25.0%, respectively). These results suggest that cryoprotectant concentrations can be reduced and still provide acceptable fertility rates.

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